Process for preparing a substrate coated with a biomolecule

ABSTRACT

Processes are provided for preparing a substrate coated with a biomolecule, comprising:
         a) providing a substrate;   b) coating the substrate with a polysiloxane, typically by exposing the substrate to a reactive gas containing siloxane functional groups and to plasma energy to yield a plasma-deposited polysiloxane surface on the substrate;   c) rendering the polysiloxane surface amino functional; and   d) contacting the amino-functional polysiloxane surface with a biomolecule under conditions effective to attach the biomolecule to the substrate.       

     The coated substrate may be a medical device that demonstrates an ability to maintain gas permeability when in contact with blood or blood components, compared to a substantially identical medical device that has not been coated with a biomolecule using this process.

The present invention claims the benefit of provisional patentapplication Ser. No. 60/909,553 entitled “Process for Preparing aSubstrate Coated with a Biomolecule” filed Apr. 2, 2007.

FIELD OF THE INVENTION

The present invention relates to processes for preparing substratescoated with biomolecules, in particular medical devices with surfacescoated with biomolecules, such as blood oxygenator fibers coated withheparin.

BACKGROUND OF THE INVENTION

Devices used in the medical field must be manufactured using materials,such as biomaterials, having particular surface properties so that thedevice functions without causing adverse effects to the patient.

Biomaterials are typically made of inert metals, polymers, or ceramicsto ensure durability and to ensure that the materials do not adverselyreact with the physiological environment with which they come intocontact, such as with blood or tissues. More particularly, manybiomedical devices may or may not require blood compatible, infectionresistant, and/or tissue compatible surfaces. For example, it is oftendesirable to manufacture medical devices, such as catheters, that haveproperties that discourage adherence of blood or tissue elements to thedevice.

It is also desirable for certain biomaterials, such as those forimplants, to be anchored stably into the tissue environment into whichthey are implanted. For example, it may be desirable for specificimplants, such as certain types of catheters and stents, to benon-inflammatory and anchored to the surrounding tissues. Moreover, itmay be desirable for certain biomaterials to prevent bacterial growthduring a course of a procedure, or as a permanent implant so as toprevent infection of a patient in contact with the biomaterial. Initialcontact of such materials with blood may result in deposition of plasmaproteins, such as albumin, fibrinogen, immunoglobulin, coagulationfactors, and complement components. The adsorption of fibrinogen ontothe surface of the material causes platelet adhesion, activation, andaggregation. Other cell adhesive proteins, such as fibronectin,vitronectin, and von Willebrand factor (vWF) also promote plateletadhesion.

In addition, disposable surgical tools may become infected with bacteriaduring a course of a long operation and reuse of the tool during theoperation may promote bacterial infection in the patient. For certaintools used in particular applications, it may be desirable therefore toprevent any bacterial growth on the surfaces of these tools during thecourse of an operation.

Additionally for permanently implanted materials it would be desirableto prevent bacterial growth that would lead to a biomaterial or devicecentered infection. In the latter the only remedy is eventual removal ofthe implant.

Adverse reactions between materials and blood components are predominantfactors limiting the use of synthetic materials that come into contactwith physiological fluids.

A number of approaches have been suggested to improve thebiocompatibility and blood compatibility of medical devices. Oneapproach has been to modify the surface of the material to preventundesirable protein adhesion by providing the material with a lowpolarity surface, a negatively charged surface, or a surface coated withbiological materials, such as enzymes, endothelial cells, and proteins.Another approach has been to bind anticoagulants to the surface ofbiologically inert materials to impart antithrombogenic characteristicsto the materials. Still another approach used in the art has been thecopolymerization of various phospholipids which are used as coatingmaterials for various substrates. Partial polymeric backbone coatingshave also been used in a similar fashion. However, many of these methodscan result in a leaching or “stripping off” of the coating.

In devices requiring the transfer of gases, for example, in bloodoxygenators requiring the exchange of oxygen and carbon dioxide througha membrane or porous fiber, there are additional drawbacks. Oftensurfaces that have been rendered biocompatible by the coating ofbiomolecules attract phospholipids. Phospholipids that adhere to thesurface coat the pores and wet the surface of the device, making ithydrophilic. Water adversely affects gas transfer, making the oxygenatorsignificantly less effective.

There is a need in the art to develop processes for preparing substratescoated biomolecules that demonstrate biocompatibility and bloodcompatibility, while maintaining gas permeability.

SUMMARY OF THE INVENTION

In accordance with the present invention, processes are provided forpreparing a substrate coated with a biomolecule. A typical processcomprises:

a) providing a substrate;

b) coating the substrate with a polysiloxane;

c) rendering the polysiloxane surface amino functional; and

d) contacting the amino-functional polysiloxane surface with abiomolecule under conditions effective to attach the biomolecule to thesubstrate.

Note that the order of some of the process steps may be altered or stepsmay be combined and performed simultaneously with the same results andwithout departing from the scope of the invention. Also, additionalsteps such as cleaning steps may be added as necessary, discussedhereinafter.

DETAILED DESCRIPTION OF THE INVENTION

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessexpressly and unequivocally limited to one referent.

For the purposes of this specification, unless otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andother parameters used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired properties to beobtained by the present invention. At the very least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

All numerical ranges herein include all numerical values and ranges ofall numerical values within the recited numerical ranges.Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The various embodiments and examples of the present invention aspresented herein are each understood to be non-limiting with respect tothe scope of the invention.

As used in the following description and claims, the following termshave the indicated meanings:

The term “cure”, “cured” or similar terms, as used in connection with acured or curable composition, e.g., a “cured composition” of somespecific description, means that at least a portion of the polymerizableand/or crosslinkable components that form the curable composition is atleast partially polymerized and/or crosslinked. For example, the degreeof crosslinking can range from 5% to 100% of complete crosslinking. Inalternate embodiments, the degree of crosslinking can range from 35% to85%, e.g., 50% to 85%, of full crosslinking. The degree of crosslinkingcan range between any combination of the previously stated values,inclusive of the recited values.

The term “curable”, as used for example in connection with a curablefilm-forming composition (coating), means that the indicated compositionis polymerizable or cross linkable, e.g., by means that include, but arenot limited to, thermal, catalytic, electron beam, chemical free-radicalinitiation, and/or photoinitiation such as by exposure to ultravioletlight or other actinic radiation.

The terms “on”, “appended to”, “affixed to”, “bonded to”, “adhered to”,or terms of like import means that the designated item, e.g., a coating,film or layer, is either directly connected to (superimposed on) theobject surface, or indirectly connected to the object surface, e.g.,through one or more other coatings, films or layers (superposed on).

The terms “attach”, “couple”, and “link” refer to securing a coating orbiomolecule to a substrate, for example, by chemical covalent or ionicbonding, such that the coating or biomolecule is immobilized withrespect to the substrate.

The term “rigid”, as used for example in connection with a substrate,means that the specified item is self-supporting.

The phrase “an at least partial film” means an amount of film coveringat least a portion, up to the complete surface of the substrate. As usedherein, a “film” may be formed by a sheeting type of material or acoating type of material. For example, a film may be an at leastpartially cured polymeric sheet or an at least partially cured polymericcoating of the material indicated. The phrase “at least partially cured”means a material in which from some to all of the curable orcross-linkable components are cured, crosslinked and/or reacted.

The term “medical device” may be a device that has surfaces that contacttissue, blood, or other bodily fluids in the course of its operation,which fluids are subsequently introduced into patients. This caninclude, for example, extracorporeal devices for use in surgery such asblood oxygenators, blood pumps, blood sensors, tubing used to carryblood and the like which contact blood which is then returned to thepatient. This can also include endoprostheses implanted in blood contactin a human or animal body such as vascular grafts, stents, pacemakerleads, heart valves, and the like that are implanted in blood vessels orin the heart. This can also include devices for temporary intravascularuse such as catheters, guide wires, and the like which are placed intothe blood vessels or the heart for purposes of monitoring or repair.

The term “biomolecule” refers to a biologically active molecule.

A “biocompatible” material does not generally cause significant adversereactions (e.g., toxic or antigenic responses) in the body, whether itdegrades within the body, remains for extended periods of time, or isexcreted whole. Ideally, a biocompatible material will not induceundesirable reactions in the body as a result of contact with bodilyfluids or tissue, such as infection, coagulation, tissue death, tumorformation, allergic reaction, foreign body reaction (rejection) orinflammatory reaction.

A “blood compatible” material is one that will not induce undesirablereactions in the body as a result of contact with blood, such as bloodclotting or infection. A blood compatible material is understood to bebiocompatible.

According to the present invention, processes are provided for preparinga substrate coated with a biomolecule. A typical process comprises:

a) providing a substrate;

b) coating the substrate with a polysiloxane;

c) rendering the polysiloxane surface amino functional; and

d) contacting the amino-functional polysiloxane surface with abiomolecule under conditions effective to attach the biomolecule to thesubstrate.

The surface of the resulting coated substrate is biocompatible andpreferably blood compatible. In particular, the resulting coatedsubstrate is permeable to oxygen and carbon dioxide and remains soduring exposure to bodily fluids by preventing adhesion ofphospholipids.

Substrates suitable for use in the process of the present inventioninclude metals, polymers, ceramic and glass. They are substantiallyinsoluble in body fluids and are generally designed and constructed tobe placed in or onto the body or to contact fluid of the body, mostoften blood. The substrates have the physical properties such asstrength, elasticity, permeability and flexibility required to functionfor their intended purpose, and are typically rigid, i.e., capable ofmaintaining their shape and supporting any subsequently-applied coatingsor films. The substrates can be purified, fabricated and sterilizedeasily; will substantially maintain their physical properties andfunction during the time that they remain implanted in or in contactwith the body or bodily fluid. Examples of such substrates include:metals such as titanium/titanium alloys, TiNi (shape memory/superelastic), aluminum oxide, platinum/platinum alloys, stainless steels,and other metal alloys known to be useful for medical devices, pyrolyticcarbon, silver or glassy carbon; polymers such as polyurethanes,polycarbonates, silicone elastomers, polyolefins including polyethylenesor polypropylenes, polyvinyl chlorides, polyethers, polyesters, nylons,polyvinyl pyrrolidones, polyacrylates and polymethacrylates such aspolymethylmethacrylate (PMMA), n-Butyl cyanoacrylate, polyvinylalcohols, polyisoprenes, rubber, cellulosics, polyvinylidene fluoride(PVDF), polytetrafluoroethylene, ethylene tetrafluoroethylene copolymer(ETFE), acrylonitrile butadiene ethylene, polyamide, polyimide, styreneacrylonitrile, and the like; minerals or ceramics such as hydroxapatite;human or animal protein or tissue such as bone, skin, teeth, collagen,laminin, elastin or fibrin; organic materials such as wood, cellulose,or compressed carbon; and other materials such as glass, or the like.

The substrate used in the process of the present invention oftencomprises a surface of a medical device. Substrates which may be coatedwith biomolecules in accordance with the present invention include, butare not limited to, those to be used in the manufacture of medicaldevices such as surgical implants, prostheses, and any artificial partor device which replaces or augments a part of a living body or comesinto contact with bodily fluids, particularly blood. The substrates canbe in any shape or form including tubular, sheet, rod and articles ofshapes required for particular uses. Such shaped substrates aretypically coated using the process of the present invention prior tomanufacture of the medical device in which they are used. Variousmedical devices and equipment usable in accordance with the inventionare known in the art. Examples of devices include catheters, suturematerial, tubing, and fiber membranes. Examples of catheters includecentral venous catheters, thoracic drain catheters, and angioplastyballoon catheters. Examples of tubing include tubing used inextracorporeal circuitry, such as whole blood oxygenators. Examples ofmembranes include polycarbonate membranes, haemodialysis membranes, andmembranes used in diagnostic or biosensor devices. Also included aredevices used in diagnosis, as well as polyester yarn suture materialsuch as polyethylene ribbon, and polypropylene hollow fiber membranes.Further illustrations of medical devices include autotransfusiondevices, blood filters, blood pumps, blood temperature monitors, bonegrowth stimulators, breathing circuit connectors, bulldog clamps,cannulae, grafts, implantible pumps, impotence and incontinenceimplants, intra-ocular lenses, leads, lead adapters, lead connectors,nasal buttons, orbital implants, cardiac insulation pads, cardiacjackets, clips, covers, dilators, dialyzers, disposable temperatureprobes, domes, drainage products, drapes, ear wicks, electrodes, embolicdevices, esophageal stethoscopes, fracture fixation devices, gloves,guide wires, hemofiltration devices, hubs, intra-arterial blood gassensors, intracardiac suction devices, intrauterine pressure devices,nasal spetal splints, nasal tampons, needles, ophthalmic devices, PAPbrushes, periodontal fiber adhesives, pessary, retention cuffs,sheeting, staples, stomach ports, surgical instruments, transducerprotectors, ureteral stents, vaginal contraceptives, valves, vesselloops, water and saline bubbles, achtabular cups, annuloplasty ring,aortic/coronary locators, artificial pancreas, batteries, bone cement,breast implants, cardiac materials, such as fabrics, felts, mesh,patches, cement spacers, cochlear implant, defibrillators, generators,orthopedic implants, pacemakers, patellar buttons, penile implants,pledgets, plugs, ports, prosthetic heart valves, sheeting, shunts,umbilical tape, valved conduits, and vascular access devices.

The method of the present invention also is particularly applicable toblood gas exchange devices, e.g., oxygenators. This includes both sheetand tubular forms of membrane oxygenators, which are well known in theart. In a membrane oxygenator, the blood is separated from directcontact with the oxygenating gas by a membrane, which is disposed withina hollow housing. This membrane is microporous or semipermeable, thatis, capable of permitting carbon dioxide and oxygen to permeate throughit while at the same time preventing the blood itself from passingtherethrough.

There currently are two types of membrane oxygenators. One type isreferred to as a hollow fiber oxygenator, and is illustrated in U.S.Pat. No. 4,239,729 (Hasegawa et al). A hollow fiber oxygenator employs alarge plurality (typically thousands) of microporous or semipermeablehollow fibers disposed within a housing. These hollow fibers are sealedin the end walls of the housing; the end walls are then fitted withskirted end caps. One end cap is fitted with an inlet, and the other isfitted with an outlet. In the Hasegawa et al. oxygenator, the hollowfibers are aligned in the housing so that their longitudinal axes aregenerally parallel to the longitudinal axis of the housing. In thisdevice, blood enters through the inlet of one end cap, passes throughthe lumens of the hollow fibers, and exits through the outlet of theother end cap. Oxygenated gas enters the device through the inlet in theperipheral wall near one end of the device, passes over the outersurfaces of the hollow fibers, and exits the device through the outletin the peripheral wall near the other end of the device. It will beunderstood that carbon dioxide diffuses from the blood flowing insidethe hollow fibers through the fiber walls into the stream of oxygenatinggas. At the same time, oxygen from the oxygenating gas flowing over theouter surfaces of the hollow fibers diffuses through the walls of thehollow fibers into the lumens thereof to oxygenate the blood flowingtherethrough.

Since the development of this type of oxygenator, other oxygenatorscomprising hollow fibers have been developed. These oxygenatorstypically comprise a plurality of hollow fibers disposed within a hollowhousing and arranged so that blood typically flows over the hollowfibers and gases typically flow through the hollow fibers. Manyconfigurations are possible as to the direction of fluid flow and thearrangement of fibers. The fibers may be in a linear, circular, orspiral arrangement, for example, or may be wrapped or wound around acore in various configurations. Hollow fiber membrane oxygenators aredescribed, for example, in U.S. Pat. No. 4,975,247 (Badolato, et al) andU.S. Pat. No. 5,395,468 (Juliar, et al). In certain embodiments of thepresent invention, the substrate being coated comprises hollow fibersthat are to be used in the manufacture of a blood oxygenator.

A second type of membrane oxygenator, called the flat plate membraneoxygenator, employs one or more thin, flat sheets of microporousmembrane. In its most basic form, the flat plate oxygenator has a singlesheet of microporous membrane sealed into a housing so as to provide inthe housing a first compartment (the “blood compartment”) for the flowof blood, and a second compartment (the “gas compartment”) for the flowof an oxygenating gas. Each of the compartments is fitted with an inletand an outlet. Blood flows into and out of the blood compartment and theoxygenating gas flows into and out of the gas compartment. Oxygen passesfrom the oxygenating gas across the membrane into the blood flowingthrough the blood compartment. Carbon dioxide passes from the enteringblood across the membrane to be entrained in the oxygenating gas. Theexiting blood, now reduced in carbon dioxide and enriched in oxygen, isreturned to the patient.

In certain embodiments of the present invention, the process may includea step of cleaning the substrate prior to step b) of the process, toremove any surface contaminants or impurities. Such cleaning may bedone, for example, by placing the substrate in a plasma chamber,infusing air, oxygen, and/or nitrogen into the plasma chamber, and thenexposing the device to plasma energy. Air and oxygen plasma treatmentsintroduce oxygen containing functionalities on the surface of polymericsubstrates. For example, hydroxyl, carboxyl, and other oxygen containingfunctionalities are introduced on the surface of polyethylene. As aresult, the surface becomes more polar and wettability increases. Lowmolecular weight contaminants are effectively removed by the combinedeffect of plasma and vacuum. Air plasma treatment of metallic substratematerials mostly provides a cleaning effect, removing hydrocarbons andother organic contaminants from the metal surface. Plasma treatment forcleaning purposes may be conducted in the same manner as reactive gastreatment, discussed below.

In step b) of the process of the present invention, the substrate iscoated with a polysiloxane. This coating step may be accomplished in anyof several manners. It is possible to contact the substrate with apolysiloxane in a liquid carrier. Contact may be by brushing, dipping(immersion), flow coating, spraying and the like. Immersion may includestirring or other agitation of the coating composition, by use of astirring device or by movement of the substrate to be coated through thecomposition. More often, however, the substrate is exposed to a reactivegas containing siloxane functional groups and plasma energy to yield aplasma-deposited polysiloxane surface on the substrate. Such plasmatreatments typically take place within a plasma chamber containingelectrodes, across which a voltage is applied, as known in the art. Astream of gas is fed into the chamber. Gases may vary and include, forexample, hexamethyldisiloxane and/or tetramethyldisiloxane. When a highfrequency voltage is applied between the electrodes, current flows intothe chamber, forming a plasma, which is a glowing electrical dischargewithin the gas. Reactive chemical species are formed in this electricaldischarge.

The plasma-deposited surface comprises a polymeric layer deposited ontothe substrate. Siloxane molecules are fragmented in the plasma phase andrecombine to yield a high molecular weight polymeric compound thatdeposits as a film on the device surface. The structure of the depositedfilm depends on the stream gas chemistry and the treatment conditions.Films deposited by this plasma process are, typically, highlycross-linked, pin-hole free, homogeneous, and show good adhesion to thedevice. Following cessation of the high frequency voltage appliedbetween the electrodes, the gas stream flow may be continued in thechamber in order to quench the substrate.

Step c) of the process of the present invention comprises rendering thepolysiloxane surface amino functional. In certain embodiments of thepresent invention, step c) comprises contacting the polysiloxane surfacewith an amino- and/or imino-functional compound for a time sufficient toeffect adsorption of the amino- and/or imino-functional compound ontothe polysiloxane surface. In such embodiments, the amino- and/orimino-functional compound may comprise polyethyleneimine, anamino-functional silane and/or diaminopropane. Examples of suitableamino functional silanes include amino-functional silanes sold as theDow Corning Z-silane series. Depending on the identity of the compound,it may be present in a liquid carrier, particularly when the compound isan amino-functional silane. Again, contact may be by brushing, dipping(immersion), flow coating, spraying and the like, but is typically byimmersion. After adsorption of the compound onto the surface, anyimino-functional groups may be reduced to amino-functional groups byaddition of a suitable reducing agent to the liquid carrier.

Alternatively, step c) may comprise exposing the plasma-depositedpolysiloxane surface to ammonia or an organic amino-functional gas andto plasma energy to yield an amino-functional plasma-deposited surface.Suitable organic amino-functional gases include amino-functionalpolysiloxane, diaminopropane, and allyl amine.

Prior to attachment of the biomolecule to the amino-functionalpolysiloxane surface in step d), it may be desirable to expose theamino-functional polysiloxane surface to a reactive gas containingacrylic acid and to plasma energy to yield a plasma-depositedpolyacrylic acid coating on the surface. This step is particularlyuseful when the polysiloxane has been applied using plasma energy, andis suitable for the preparation of medical devices that do notfacilitate mass transfer such as gas exchange.

In step d) of the process of the present invention, the amino-functionalpolysiloxane surface is contacted with a biomolecule under conditionseffective to attach the biomolecule to the substrate. Examples ofbiomolecules that may be attached to the surface include antibacterialagents, antimicrobial agents, anticoagulants, antithrombotic agents,platelet agents, anti-inflammatories, enzymes, catalysts, hormones,growth factors, drugs, vitamins, antibodies, antigens, nucleic acids,dyes, a DNA segment, an RNA segment, protein, and peptides. Often, whenthe medical device to be coated is designed to come in contact withblood, in particular when the medical device is a blood oxygenator, thebiomolecule comprises heparin.

Attachment of the biomolecule to the amino-functional polysiloxanesurface can be accomplished by any of a number of methods known to thoseskilled in the art. One particularly preferred method is an oxidationmethod involving the use of periodate. The biomolecule, usually heparin,is contacted with a periodate in a buffered aqueous solution and allowedto react. This controlled oxidation provides a limited number ofreactive aldehyde groups per molecule. The periodate is a water-solubleperiodate, preferably, an alkali metal periodate, such as sodiumperiodate. When the biomolecule is heparin, the amount of periodate usedis sufficient to react with no more than two of the sugar units in theheparin molecule (i.e., the basic disaccharide residues constituting thestructure of the glycosaminoglycan). If the periodate used is sodiumperiodate and the heparin used is a commercially available injectableform of heparin (e.g., its sodium salt with activity of 160units/milligram), the weight ratio of heparin to periodate should beabout 30:1 or less in order to react with no more than two of the sugarunits in the heparin molecule. It will be appreciated by those skilledin the art that the amount of periodate required for other periodatecompounds and other forms of heparin can be determined by conventionalcalculation and empirical tests.

The reaction between heparin and periodate takes place in an aqueousbuffer solution. Generally, buffers having a pH in a neutral to slightlyacidic range of about 4.5 to about 8 can be used. A lower pH (e.g., anacetate buffer at pH 4.5) is preferred if a rapid reaction is desiredwhile a more neutral pH (e.g., a phosphate buffer at pH 6.88) ispreferred for a slower reaction with a longer storage life. With theacetate buffer at a pH of 4.5, the reaction should proceed for about 3hours, while with a phosphate buffer at a pH or 6.88, the reactionshould proceed for about 16 hours. If desired, the reacted mixture maythen be stored prior to use at about 5° C.

The reacted mixture is diluted and the pH adjusted in order to bring thepH of the mixture to a pH that is favorable for the coupling reactionbetween the biomolecule and the amino-functional polysiloxane. A mildreducing agent, such as sodium cyanoborohydride, is added to the dilutedmixture to effect the reduction of the bonds formed between the reactivealdehyde groups on the oxidized biomolecule and the amine functionalgroups on the polysiloxane coated on the substrate surface. Thesubstrate surface being treated is then contacted with (e.g., immersedin or flushed with) the diluted mixture at a sufficient temperature andfor a sufficient time to complete the reaction (i.e., attach thebiomolecule). This time can range from about 30 seconds to about 2 hoursat temperatures ranging from about 20° C. to about 60° C. For example,at room temperature (i.e., about 20° C. to about 25° C.), the substratecoated with the amino-functional polydimethylsiloxane can be flushedwith a solution of a biomolecule over a period of 30 seconds to 5minutes for effective biomolecule attachment.

Substrates coated with biomolecules according to the process of thepresent invention are biocompatible, and are typically blood compatible,while remaining permeable to gases including oxygen and carbon dioxide.

The present invention is more particularly described in the followingexamples, which are intended to be illustrative only, since numerousmodifications and variations therein will be apparent to those skilledin the art. Unless otherwise specified, all parts and percentages are byweight.

EXAMPLES

One group of modified bulk material was prepared, a total of 34 hollowfiber strips underwent a 40 sec O₂/N₂ plasma cleaning followed by a40-second siloxane deposition. Within 48 to 72 hours the siloxanetreated material was heparinized. (NH)

Materials and Methods

-   -   1. Microporous Hollow Fiber Membrane Bulk Material Lot#        13502-4-4 precut to 36″ lengths    -   2. Glass microscope slide    -   3. Siloxane-Tetramethyldisiloxane, 97% P/N 235733//Batch 04526KC        (Aldrich)    -   4. For chemical list see table IV        Set-Up and Pre-Testing    -   1. Glass microscope slide    -   2. After placing the glass slide in the reactor and pulling        vacuum to <100 mtorr, oxygen was allowed through the mass flow        controller (MFC1) at a rate of 20% and N₂ through MFC2 at a rate        of 80% of total flow, and the pressure control was set to 250        motor. Plasma power was set for 200 W (power). See table I below        Pre-Test for Uniformity of Siloxane Deposition on Glass Slide

TABLE I Set-Up Parameters Siloxane Set Temperature Material ProcessPressure Power Time Set Description Description (mtorr) MFC1/MFC2 WattsSeconds Point Comments Glass 20% O₂ 250 0.2/.8 200 40 NA Contactmicroscope 80% N₂ angle 0 - slide and Clean wet out PP HF Siloxane 250NA 200 40 NA Contact Deposition angle >90 - non wetSet-Up Procedure

Siloxane vapors from a feed chamber were introduced through a ball valvethat communicated with the plasma reactor. Vacuum was pulled to <100mtorr before opening the ball valve. The valve was opened to controlpressure at 250 mtorr from the siloxane vapor.

Results from first test for uniform coverage in the reactor showed thatthe glass slide made the conversion from hydrophilic to hydrophobic.

Siloxane Deposition of Bulk Material

-   -   1. Two 36″ hollow fiber strips per/run were placed on the        reactor tray and carefully taped underneath. See diagram below    -   2. O₂//N₂ cleaned for 40 seconds    -   3. Siloxane deposition 40 seconds    -   4. Contact angle was performed on a glass microscope slide after        each siloxane treatment/run.    -   5. For storage and transporting after siloxane deposition, the        strips were placed between lint-free towels.

TABLE II Set-Up Parameters Siloxane Contact Material Process RunPressure MFC1/ Set Power Time Temperature Angle Description DescriptionNumber (mtorr) MFC2 Watts Seconds Set Point Glass Slide (2) 36 × 4″O₂//N₂ Clean 1 250 0.2 200 40 NA 0 - wet Strips out (1) Glass Siloxane0.8 40 >90 - non Slide wet (2) 36 × 4″ O₂//N₂ Clean 2 250 0.2 200 40 NA0 - wet Strips out (1) Glass Siloxane 0.8 40 >90 - non Slide wet (2) 36× 4″ O₂//N₂ Clean 3 250 0.2 200 40 NA 0 - wet Strips out (1) GlassSiloxane 0.8 40 >90 - non Slide wet (2) 36 × 4″ O₂//N₂ Clean 4 250 0.2200 40 NA 0 - wet Strips out (1) Glass Siloxane 0.8 40 >90 - non Slidewet (2) 36 × 4″ O₂//N₂ Clean 5 250 0.2 200 40 NA 0 - wet Strips out (1)Glass Siloxane 0.8 40 >90 - non Slide wet (2) 36 × 4″ O₂//N₂ Clean 6 2500.2 200 40 NA 0 - wet Strips out (1) Glass Siloxane 0.8 40 >90 - nonSlide wet (2) 36 × 4″ O₂//N₂ Clean 7 250 0.2 200 40 NA 0 - wet Stripsout (1) Glass Siloxane 0.8 40 >90 - non Slide wet (2) 36 × 4″ O₂//N₂Clean 8 250 0.2 200 40 NA 0 - wet Strips out (1) Glass Siloxane 0.840 >90 - non Slide wetWet Chemistries (PEI and Heparin)

34 siloxane treated bulk material sheets (pre-cut to ˜17×4″) thencarefully layered into (2) vessels and modified as follows.

Step One—Polyethyleneimine (PEI) Amination:

Preparation of BASF PEI solution [0.1%]: Total=1800 g

1764 g 0.1M Borate Buffer pH 9.0

36 ml of a 5% BASF PEI stock solution

PEI and borate buffer were combined a glass beaker and allowed to stirfor 15 minutes, the PEI solution was dispensed into (2) 2000 mlrectangle vessels each containing 16 (17×4″) layered material strips.The container's were covered and placed on an orbital shaker and allowedto agitate @90 rpm for 75 minutes @ ambient temperature. After PEIadsorption, the aminated material was rinsed several times with DI H₂O.After rinsing a small sample was removed, stained with Ponceau S andevaluated for uniformity.

Ponceau S Staining Results:

The aminated sample showed a light uniform pink stain, indicatinguniform coverage of PEI.

Step Two—Heparinization:

Preparation of Deaminated Heparin (DH) solution Total=1800 g

1.8 g DH heparin = [1 mg/ml] 1800 g 0.5M NaCl adjust to pH 4.0 0.18 gNaCNBH₃ = [0.1 mg/ml]

Heparin was dissolved in the pre-mixed NaCl solution, then adjusted topH 4.0±0.1, the solution was then preheated to 55° C. After the solutionreached temperature the NaBHCN₃ was added and allowed to mix for 5-10minutes. The preheated heparin solution was dispensed into (2) 2000 mlrectangle vessel containing the aminated material, the container's werecovered and placed in a pre-heated 55° C. Orbital shaker @90 rpm for 2hours 55° C. After heparinization the modified material was rinsed withDI H₂O, 1M NaCl, followed with a final DI rinse. After rinsing a smallsection was removed, stained with Toluidine Blue and evaluated foruniformity.

Toluidine Blue O Staining Results:

Visual observations showed the heparinized sample to have a light butuniform purple stain, indicating uniform coverage of heparin.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims and equivalents thereto.

What is claimed is:
 1. A process for preparing a substrate coated with abiomolecule comprising: a) providing a substrate; b) coating thesubstrate with a polysiloxane by exposing the substrate to a reactivegas containing siloxane functional groups and to plasma energy to yielda plasma-deposited polysiloxane surface on the substrate; c) renderingthe polysiloxane surface amino functional after the step of coating thesubstrate with a polysiloxane; and d) contacting the amino-functionalpolysiloxane surface with a biomolecule under conditions effective toattach the biomolecule to the substrate by a reaction between thebiomolecule and the amino-functional polysiloxane.
 2. The process ofclaim 1 wherein the substrate is a metal, polymer, ceramic, or glass. 3.The process of claim 2 wherein the substrate is a surface of an articleto be used in the manufacture of a medical device.
 4. The process ofclaim 3 wherein the medical device is a blood oxygenator.
 5. The methodof claim 4 wherein the medical device is a blood oxygenator and thesubstrate being coated comprises hollow fibers.
 6. The process of claim1 wherein the reactive gas containing siloxane functional groups isselected from the group consisting of hexamethyldisiloxane andtetramethyldisiloxane.
 7. The process of claim 1 wherein step c)comprises contacting the polysiloxane surface with an amino- and/orimino-functional compound for a time sufficient to effect adsorption ofthe amino- and/or imino-functional compound onto the polysiloxanesurface.
 8. The process of claim 7 wherein the amino- and/orimino-functional compound comprises polyethyleneimine, anamino-functional silane and/or diaminopropane.
 9. The process of claim 1wherein step c) comprises exposing the plasma-deposited polysiloxanesurface to ammonia or an organic amino-functional gas and to plasmaenergy to yield an amino-functional plasma-deposited surface.
 10. Theprocess of claim 9 wherein the plasma-deposited polysiloxane surface isexposed to an organic amino-functional gas selected fromamino-functional polysiloxane, diaminopropane, and allyl amine.
 11. Theprocess of claim 1 wherein step d) comprises contacting theamino-functional polysiloxane surface with a biomolecule in a liquidcarrier at a temperature of at least 20° C. for at least 30 seconds. 12.The process of claim 11 wherein the liquid carrier further comprises aperiodate in a buffered aqueous solution.
 13. The process of claim 12wherein the periodate comprises an alkali metal periodate.
 14. Theprocess of claim 12 wherein the biomolecule comprises heparin and theperiodate is present in a sufficient amount to form aldehyde groups onthe heparin.
 15. The process of claim 12 wherein the buffered aqueoussolution has a pH in a range of about 4.5 to about
 8. 16. The process ofclaim 1 wherein the biomolecule is selected from the group consisting ofan antibacterial agent, an antimicrobial agent, an anticoagulant, anantithrombotic agent, a platelet agent, an anti-inflammatory compound,an enzyme, a catalyst, a hormone, a growth factor, a drug, a vitamin, anantibody, an antigen, a nucleic acid, a dye, a DNA segment, an RNAsegment, a protein, and a peptide.
 17. The process of claim 1, furthercomprising, prior to step b), a step of placing the substrate in aplasma chamber, infusing air, oxygen, and/or nitrogen into the plasmachamber, and then exposing the device to plasma energy to clean thesubstrate surface.
 18. The process of claim 1 wherein the surface formedis biocompatible.
 19. The process of claim 18 wherein the surface formedis blood compatible.
 20. A process for preparing a substrate coated witha biomolecule comprising: a) providing a substrate; b) coating thesubstrate with a polysiloxane; c) rendering the polysiloxane surfaceamino functional after coating the substrate with a polysiloxane in stepb); and d) contacting the amino-functional polysiloxane surface with abiomolecule under conditions effective to attach the biomolecule to thesubstrate by a reaction between the biomolecule and the amino-functionalpolysiloxane.